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  • 1
    Electronic Resource
    Electronic Resource
    Isolation and characterisation of a 13.8-kDa bacteriolytic enzyme from house dust mite extracts: homology with prokaryotic proteins suggests that the enzyme could be bacterially derived (2002)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS immunology and medical microbiology 33 (2002), S. 0 
    Details
    ISSN: 1574-695X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Bacteriolytic activity was detected in extracts of whole mite and spent growth medium (SGM) from the clinically important Dermatophagoides pteronyssinus and Dermatophagoides farinae mites and was most abundant in whole mite extract. Gram-positive organisms Micrococcus lysodeikticus, Bacillus megaterium and Listeria monocytogenes were preferentially lysed and the lytic activity was enhanced by thiols, destroyed by mite proteases, inhibited by HgCl2 and high concentrations of NaCl but was resistant to heat and acid treatment. Substrate SDS–PAGE analysis indicated the presence of several lytic enzymes, two of which were isolated from D. pteronyssinus spent growth medium extract by hydroxyapatite chromatography. The N-terminal amino acid sequence of one of them was then used in PCR-based cloning studies. The complete amino acid sequence of this protein was determined and cDNA found to encode a 130-amino acid residue mature protein with a 20-amino acid leader sequence. The deduced protein demonstrated sequence similarity with the C-terminal regions of a group of bacterial proteins belonging to the P60 superfamily. These data suggest that the enzyme is derived from bacteria within the mites rather than from mites per se.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-695X.2002.tb00576.x
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  • 2
    Electronic Resource
    Electronic Resource
    The use of capillary liquid chromatography and 2D-gel electrophoresis for isolating proteins for high-sensitivity protein sequence analysis (1992)
    Springer
    The protein journal 11 (1992), S. 352-353 
    Details
    ISSN: 1573-4943
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01673702
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  • 3
    Electronic Resource
    Electronic Resource
    Fabrication of Stable Packed Capillary Reversed-Phase Columns for Protein Structural Analysis (1997)
    Springer
    The protein journal 16 (1997), S. 425-431 
    Details
    ISSN: 1573-4943
    Keywords: Reversed-phase microcolumn liquid chromatography ; protein concentration ; column end-frit fabrication ; phenylthiohydantoin amino acid analysis ; capillary chromatography
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Capillary column (≤320-μm ID) liquid chromatography is an essential tool for the separation and concentration of low-picomole amounts of proteins and peptides for mass-spectrometric based structural analysis. We describe a detailed procedure for the fabrication of stable and efficient 50- to 180-μm ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reversed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-silica particles were employed for column end-frit construction. The ability of these columns to withstand high back pressures (300–400 bar) enabled their use for rapid chromatography (〉3400 cm/hr; i.e., ∼40 μl/min for 200-μm ID columns) and the loading of large sample volumes (up to 500 μl). The accurate low flow rates (0.4–4.0 μl/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr. 599, 119–130]. Column performance was evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1026345023941
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  • 4
    Electronic Resource
    Electronic Resource
    S-Pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: A rapid mass spectrometric method for identifying cysteine-containing peptides (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 907-917 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional gel polyacrylamide gel electrophoresis ; Mass spectrometry ; Peptide mass fingerprinting ; Capillary liquid chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In-gel proteolytic digestion of acrylamide-gel separated proteins is a method widely used for generating peptide fragments for the purpose of identifying proteins by Edman degratation, tandem mass spectrometry, and peptide-mass fingerprinting. However, it is well recognised for disulfide-bonded proteins electrophoresed under reducing conditions that if no precautions are taken to minimise disulfide bond formation during protein digestion or peptide isolation, complex peptide maps can result. Here, we describe an improved method for in-gel protein digestion. It consists of first reducing and S-pyridylethylating Coomassie Brilliant Blue R-250-stained proteins immobilised in the whole gel slab with dithiothreitol and 4-vinylpyridine, excising the individual stained and alkylated proteins, and then digesting them in situ in the gel matrix with trypsin or Achromobacter lyticus protease I. Peptide fragments generated in this manner are extracted from the gel piece and purified to homogeneity by a rapid (≤12 min) reversed-phase high performance liquid chromatography (HPLC) procedure, based upon conventional silica supports. Recoveries of peptides are increased by S-pyridylethylation of acrylamide-immbilised proteins prior to in-gel digestion. Further, the levels of gel-related contaminants, which otherwise result in suppression of sample signals during electrosprayionisation mass spectrometry, are greatly reduced by the reduction / alkylation step. Additionally, we demonstrate that S-β-(4-pyridylethyl)-cysteine containing peptides can be readily identified during reversed-phase HPLC by absorance at 254 nm, and during electrospray ionisation tandem mass spectrometry by the appearance of a characteristic-pyridylethyl fragment ion of 106 Da. The position of cysteine residues in a sequence can be determined as phenylthiohydantoin S-β-(4-pyridylethyl)-cysteine during Edman degradation, and by tandem mass spectrometry.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170512
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  • 5
    Electronic Resource
    Electronic Resource
    Two-dimensional electrophoretic analysis of proteins expressed by normal and cancerous human crypts: Application of mass spectrometry to peptide-mass fingerprinting (1994)
    Weinheim : Wiley-Blackwell
    Electrophoresis 15 (1994), S. 391-405 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Protein patterns of normal human colonic crypts, isolated from different regions of the large intestine, and several colorectal cancer cell lines were compared using two-dimensional electrophoresis gels (2-DE). As detected by intrinsic radiolabeling and Coomassie Brilliant Blue staining, the protein patterns for normal crypts isolated from the ascending, and descending, regions of the colon and the rectum, were almost (〉 95%) identical. While 75-80% of the protein spots from normal crypts and the colorectal cancer cell line (LIM 1863), a cell line that grows as organoids and differentiates spontaneously into crypt-like structures in vitro, can be matched, the relative expression levels of a large number of proteins differ. At least two protein spots (undetectable in the protein pattern from normal cells), proteins a (Mr ∼ 18 000, pI 6.7-6.9) and b (Mr ∼ 24 000, pI 5.9-6.0), were detected in the 2-DE gel protein pattern in the three cell lines LIM 1863, LIM 1215 and LIM 1899. The identity of these proteins is not yet known and further studies are required before they can be considered as potential colon tumor markers. Approximately 60% of the cellular proteins from LIM 1215 cells, a colon carcinoma cell line that exhibits many properties associated with columnar cells, can be matched with LIM 1863 cells. The results presented here represent an initial phase in our efforts to develop a comprehensive protein database for normal human colon cells and several colorectal cancer cell lines. While our initial protein identification relied on microsequencing methodologies, we are presently evaluating peptide-mass fingerprinting, utilizing capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry, as a means for rapid identification of proteins at subpicomole levels. Using this approach, protein #3 (Mr ∼ 66 000, pI 6.2) was identified as heat shock protein 60 from as few as seven tryptic peptide masses when they were screened against the molecular weight search (MOWSE) peptide-mass database.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150150158
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  • 6
    Electronic Resource
    Electronic Resource
    Nonreducing two-dimensional polyacrylamide gel electrophoretic analysis of human colonic proteins (1995)
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 1120-1130 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Mass spectrometry ; Peptide mass fingerprinting ; Capillary liquid chromatography ; Human colonic proteins ; Immunoblot analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Immunochemical detection of proteins with antigenic determinants that are dependent on the native spatial conformation of the protein can often pose problems with conventional two-dimensional polyacrylamide gel electrophoresis (2-DE). For example, many antigenic determinants are readily destroyed by reducing agents and/or urea, reagents which are critical components of many of the conventional isoelectric focusing and immobilized-pH-gradient (IPG) protocols used in the first electrophoretic dimension. Here we describe the use of commercially available precast 2-DE gels for performing nonreducing/non-urea 2-DE of proteins extracted from the human colon cancer cell line LIM 1215 with 0.3% Triton X-100 that permit the identification of antigens with conformational determinants by immunoblot analysis. Previous, related studies demonstrated the usefulness of peptide-mass fingerprinting for identifying 2-DE resolved proteins. Here we show how partial protein sequence data obtained by rapid peptide mapping, using capillary column liquid chromatography directly coupled with electrospray ionization tandem mass spectrometric methodologies, enhances the usefulness of this approach for identifying incompletely resolved proteins. The nonreducing 2-DE gel images reported in this study, along with our master 2-DE gel protein database for both normal human colonic crypts and several colon-cancer-derived cell lines, and information regarding microtechniques employed in this laboratory for obtaining structural data on 2-DE resolved proteins can be accessed over the Internet using World Wide Web (URL address: http://www.ludwig.edu.au).
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501601189
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  • 7
    Electronic Resource
    Electronic Resource
    Purification of proteins and peptides for sequence analysis using microcolumn liquid chromatography (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Microcolumn Separations 4 (1992), S. 485-489 
    Details
    ISSN: 1040-7685
    Keywords: reversed phase microcolumn liquid chromatography ; peptide mapping ; protein microsequencing ; capillary chromatography ; protein concentration ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Reversed phase microcolumn (0.32 mm i.d.) liquid chromatography was used to purify low-picomole amounts of proteins and peptides suitable for structural analysis. Using this approach, rapid trace enrichment (concentration) of low nanogram levels of proteins from volumes as high as 500 μL down to 1-2 μL was demonstrated. The total system recovery (including manual collection and reinjection) for 50 ng of lysozyme was 〉95%; the overall recovery after five injections was 90%. Using the same packing, Brownlee RP-300, the resolution of a standard mixture of proteins was comparable for columns varying in length from 250 mm to 10mm. Identification by amino acid sequence analysis of selected peptides recovered from a sub-10 pmol Staphylococcus aureus V8 protease digest of recombinant murine interleukin-6 (IL-6) was achieved.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mcs.1220040604
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  • 8
    Electronic Resource
    Electronic Resource
    A two-dimensional gel database of human colon carcinoma proteins (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 605-613 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Tandem mass spectrometry ; Peptide mass fingerprinting ; Post-source-decay fragmentation ; Human colonic proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The master two-dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883-891; Ji et al., Electrophoresis 1994, 15, 391-405). Updated two-dimensional electrophoretic (2-DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2-DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their Mr/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2-DE resolved proteins. This procedure relies on a combination of capillary column (0.10-0.32 mm internal diameter) reversed-phase HPLC peptide mapping of in-gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision-induced dissociation or post-source-decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro-techniques employed in this laboratory for identifying 2-DE resolved proteins can be accessed via the internet URL: http://www.ludwig.edu.au.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180344
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  • 9
    Electronic Resource
    Electronic Resource
    Two-dimensional electrophoretic analysis of human breast carcinoma proteins: Mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2 (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 588-598 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional gel protein database ; Human breast carcinoma proteins ; Protein kinases ; Signal transduction components ; SH3 binding proteins ; Mixed lineage kinases ; Tandem mass spectrometry ; Protein identification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (Eur. J. Biochem. 1993, 213, 701-710), comprises several distinct structural domains including an src homology-3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif. Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction. To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23-77), fused to glutathione S-transferase. This fusion protein was expressed in Escherichia coli, purified using gluthathione-Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2-SH3 domain binding proteins from lysates of 35S-labelled MDA-MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low-abundance proteins with a molecular weights (Mr) of ˜ 31 500 and ˜ 34 000, bound consistently to the MLK2N protein. To establish accurately the Mr / isoelectric point (pI) loci of these MLK2-SH3 domain binding proteins, a number of abundant proteins in a two-dimensional electrophoresis (2-DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in-gel proteolysis and fractionation by rapid (˜ 12 min)˜ microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide-mass fingerprinting and electrospray (ESI) - mass spectrometry (MS)-MS utilizing capillary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2-DE protein database for the human breast carcinoma cell line MDA-MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: www.ludwig.edu.au/www/jpsl/jpslhome.html).
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180342
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  • 10
    Electronic Resource
    Electronic Resource
    Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33 (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 614-621 
    Details
    ISSN: 0173-0835
    Keywords: Monoclonal antibody A33 ; Two-dimensional polyacrylamide gel electrophoresis ; Tandem mass spectrometry ; Peptide mass fingerprinting ; Human colonic proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The murine monoclonal antibody A33 (mAbA33) recognises a human cell membrane-associated antigen selectively expressed in epithelial cells of the lower gastrointestinal tract and 〉 90% of colonic cancers, but is not detected in a wide range of other normal tissues by immunohistochemical analysis. In phase I/II clinical triasl, mAbA33 has been shown to target advanced colon cancers and the humanised version is currently being evaluated in therapy studies. Although the mAbA33 has been well characterised by immunohistochemical and clinical studies, until recently, the target antigen has remained poorly defined. This was largely attributable to the antigenic determinant recognised by mAbA33 being dependent on the native spatial conformation of the A33 antigen which impeded its identification by conventional two-dimensional electrophoresis (2-DE) and immunoblot analysis. We have developed an immunoblot method, based on nonreducing/non-urea precast 2-DE gels, that has facilitated the purification of the detergent (0.3% Triton X-100) solubilised A33 antigen from the human colon cancer cell lines LIM1215 and SW1222. Under these 2-DE conditions, the A33 antigen electrophoreses with an apparent Mr ˜ 41000 and pI 5.0-6.0. Attempts to isolate the A33 antigen from 2-DE gels for direct structural analysis were unsuccessful, due to its co-electrophoresis with actin and cytokeratin proteins. However, using Western blot and biosensor detection the A33 antigen has been purified chromatographically and N-terminal sequence analysis was possible. Using polyclonal antibodies raised against a synthetic peptide corresponding to the N-terminal region of the A33 antigen we have used Western blot analysis to localise the molecule in our master 2-DE protein database for normal human colon crypts and several colon carcinoma cell lines (URL address: www.ludwig.edu.au). Under reducing 2-DE conditions, the A33 antigen electrophoresis as 6 differentially charged isoforms (pI 4.6-4.8) with a single molecular weight species at Mr ˜ 55000.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180345
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